title: Cellular recovery of glyceraldehyde-3-phosphate dehydrogenase activity and thiol status after exposure to hydroperoxides.
abstract: The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.
authors.affiliation: Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
authors.forename: A E
authors.initials: AE
authors.lastname: Brodie
authors.forename: D J
authors.initials: DJ
authors.lastname: Reed
mesh_terms: D002460:Cell Line; D003543:Cysteamine / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D003553:Cystine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004976:Ethacrynic Acid / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D019803:Glutathione Disulfide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008175:Lung Neoplasms; D010084:Oxidation-Reduction; D010545:Peroxides / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014465:Ultrasonics; D020122:tert-Butylhydroperoxide
chemical_list: D010545:Peroxides; D013438:Sulfhydryl Compounds; D003553:Cystine; D003543:Cysteamine; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione; D003545:Cysteine; D004976:Ethacrynic Acid; D005033:Ethylmaleimide; D004229:Dithiothreitol; D019803:Glutathione Disulfide